According to the literature, urokinase-type plasminogen activator (u-PA) has been found in all mammalian species so far investigated. Several findings relate u-PA to tissue degradation and/or cell migration, presumably through a breakdown of the extracellular matrix, caused by plasmin together with other proteolytic enzymes (see Dan.o slashed. et al., 1988, 1990, Grondal-Hansen et al., 1988, Andreasen et al, 1990).
Immunocytochemical studies have suggested that in the invasive areas of tumors, u-PA is located at the membrane of the tumor cells (Skriver et al., 1984), and recent findings indicate that at cell surfaces, u-PA is generally bound to a specific receptor and that this localization may be crucial for the regulation of u-PA catalyzed plasminogen activation in time and space (see Blasi et al., 1987, Dan.o slashed. et al, 1990).
Surface Receptor for U-PA
The cellular receptor for u-PA (u-PAR) was originally identified in blood monocytes and in the monocyte-like U937 cell line (Vassalli et al., 1985), and its presence has been demonstrated on a variety of cultured cells, including several types of malignant cells, human fibroblasts, and also in human breast carcinoma tissue. The receptor binds active 54 kD u-PA, its one-polypeptide chain proenzyme, pro-u-PA, as well as 54 kD u-PA inhibited by the active site reagent DFP, but shows no binding of the low molecular weight (33 kD) form of active u-PA (Vassalli et al., 1985; Cubellis et al., 1986). Thus, binding to the receptor does not require the catalytic site of u-PA, and in agreement with these findings, the binding determinant of u-PA has been identified in the amino-terminal part of the enzyme, in a region which in the primary structure is remote from the catalytic site. The receptor binding domain has been established to be located in the 15 kD amino-terminal fragment (ATF, residues 1-135) of the u-PA molecule, more precisely within the cysteine-rich region termed the growth factor region as this region shows homologies to the part of epidermal growth factor (EGF) which is responsible for binding to the EGF receptor. The amino acid residues which appear to be critical for binding are located within the sequence 12-32 (Appella et al., 1987). Synthetic peptides have been constructed that inhibit the binding at very low (100 nM) concentrations. The lack of cross-reactivity between the murine and the human peptides indicates that the binding between u-PA and u-PAR is strongly species specific.
The human u-PA receptor has been purified and characterized (Behrendt et al. 1990) and its full length cDNA has been cloned (Roldan et al, 1990). The cDNA for uPAR encodes a 335 residue polypeptide which after removal of the signal sequence (Roldan et al., 1990) is further truncated during the post-translational removal of a COOH-terminal signal peptide responsible for the addition of a glycolipid membrane anchor (Ploug et al., 1991a). The mature uPAR sequence (residues 1-283) is divided into three cysteine-rich repeats of approximately 90 amino acids covering the entire sequence and it has therefore been proposed that uPAR is composed of three homologous domains (Behrendt et al., 1991, Ploug et al., 1991b).
These internal repeats of uPAR appear to be related to a family of single domain, glycolipid-anchored membrane glycoproteins, which includes the membrane inhibitor of reactive lysis (MIRL/CD59) and the murine Ly-6 antigens (Palfree 1991, Ploug et al., 1991b). Recently, the disulfide bond connectivity of the NH.sub.2 -terminal domain of uPAR has been solved (Ploug et al., 1993) and has been found to be homologous to that of the non-glycosylated snake venom .alpha.-neurotoxins suggesting that the individual UPAR domains adopt the same overall structural topology as these toxins.
The interaction between u-PA and u-PAR is entirely governed by the high receptor-binding affinity of the small epidermal growth-factor like module of uPA. It has been shown previously that the NH.sub.2 -terminal domain of u-PAR (residues 1-87; domain 1) associates with u-PA since, firstly, a covalently cross-linked adduct between this domain and u-PA can be formed selectively using disuccinimidyl suberate (Behrendt et al., 1991) and secondly, a monoclonal antibody reactive with this domain inhibits uPA binding to cells (R.o slashed.nne et al., 1991).
WO 92/07083 discloses i.a. a monoclonal antibody (as well as its use as a drug and its use in methods for the targeting of drugs) which strongly inhibits cell surface plasminogen activation, while this activation is not or only slightly affected by three other antibodies. The monoclonal antibody (3R) also efficiently inhibited binding of radiolabelled DFP-treated u-PA on the surface of U937 cells, while no or only slight inhibition was seen with the three. As the binding of the 3R antibody was completely inhibited by pre-treatment with u-PA it was concluded that this antibody bound to an epitope in the u-PA binding domain of u-PAR.
Based upon these findings, it was in WO 92/07083 concluded that inhibition of receptor binding of u-PA is a means of inhibiting some of its physiological functions in relationship to therapeutic prevention of localized proteolytic activity, e.g. invasion and metastasis of cancer cells, inflammatory bowel disease, premalignant colonic adenomas, septic arthritis, osteoarthritis, rheumatoid arthritis (for which a direct involvement of excess u-PA production has been demonstrated), osteoporosis, cholesteatoma, and a number of skin and corneal diseases for which an excess plasminogen activation has been shown to be the pathogenetic cause, such as corneal ulcers, keratitis, epidermolysis bullosa, psoriasis, and pemphigus. Since u-PA receptors are present on several blood cells (neutrophilic granulocytes and monocytes) and endothelial cells, their regulation might also significantly affect intravascular fibrinolytic activity in physiological, pathological and pharmacological conditions. The above-mentioned diseases would be the first targets for a therapy based on administration of substances that block or decrease cell surface plasminogen activation. Because of a role of u-PA in implantation of the fertilized egg, a contraceptive effect is expected of measures that inhibit receptor binding. The therapy and prophylaxis would involve systemic or topical treatment with agents that block or reduce receptor bound plasminogen activator activity.
WO 92/07083 also discloses that the u-PA binding part of u-PAR is located within the first 87 N-terminal amino acids, and the monoclonal antibodies of WO 92/07083 were shown to bind to the ligand binding part of u-PAR.